Splicing of intervening sequences from precursor-tRNA requires at least two functions, an endonuclease and a ligase. These enzymes catalyze excision of the introns and rejoining of the tRNA half-molecules through recognition of conserved features of tRNA structure. Despite the fact that tRNA splicing is readily amenable to genetic and biochemical analysis, little is known about the regulatory and structural genes involved, the potential interaction of proteins or other components that might form a splicing complex, or the possible relationship between splicing and transport of tRNA from nucleus to cytoplasm. Information on these aspects of tRNA splicing have not been forthcoming due to the intractable nature of the membrane-associated tRNA-splicing endonuclease. Previous attempts in several laboratories to study the endonuclease by either forward or reverse genetic techniques have not been successful. Recently, however, mutations in SEN (splicing endonuclease) genes were identified in this laboratory that cause partial deficiencies of endonuclease activity in the yeast Saccharomyces cerevisiae. One mutant also causes pre- tRNA steady-state accumulation and temperature-sensitive growth. By analyzing SEN genes and their products using genetic, biochemical, and immunological approaches the research in this proposal will provide critical information on the structure, organization, function, and intracellular location of tRNA splicing components. The most immediate question to be answered centers on which products are structural components of the endonuclease. Once this central question is answered it should be possible to address the more complex issues of how and where tRNA splicing occurs in intact cells.